Mycoplasma hominis strain 1620 was isolated in 1984 from the synovial exudates of a patient with severe recurring septic arthritis. Since the first isolation, multiple isolates have been obtained. Variation in size and number of surface-exposed proteins of the M. hominis patient isolates was previously reported. We analyzed the methylation of the DNAs from 14 of these patient isolates with restriction enzyme isoschizomers and nearest neighbor analysis in order to determine if the protein variation could be associated with methylation at the DNA level. Many prokaryotes, including many mycoplasmas, methylate adenine, while M. hyorhinis methylates both adenine and cytosine. All the M. hominis isolates from this patient were found to methylate cytosine. The nearest neighbor analysis experiments demonstrated that methylated cytosines occur 5' to all four bases. However, in contrast to the observed protein variations, no differences in the state of methylation among the patient M. hominis isolate DNAs were seen. Furthermore, strains of M. hominis from other patients were analyzed and could be categorized into four groups based on their methylation of bases. This observation suggests the existence of strain differences of methylases in this mycoplasma.